The subject of the invention is a medicine for treating apoptosis dysfunctions.
The term  less than  less than apoptosis greater than  greater than  is intended to refer to programmed cell death or cell suicide.
This death corresponds to a self-elimination of cells according to a defined program.
It reveals itself, initially, through bulges in the plasma membrane, these bulges being accompanied by a structural change in the membrane, and then through a loss of volume of the cell, which appears to contract and to collapse in on itself.
The nucleus condenses and the DNA is cleaved into small fragments (Raff,  less than  less than Nature greater than  greater than , 356, 397, 1992; Bortner et al.,  less than  less than Trends in Cell. Biol. greater than  greater than  5, 21, 1995).
In vivo, the cell undergoing apoptosis is recognized by macrophages which will phagocytose it and eliminate it without any inflammatory process.
Still in vivo, apoptosis is widely used by living organisms to control cell populations, in particular lymphocytes subsequent to their activation.
Moreover, during the development of organisms, apoptosis plays a fundamental role in the elimination of unnecessary embryonic tissues (lizard tail, rudiment of the genital organs of one sex or the other) and in the pattern of the organism (elimination of the interdigital webs between the future fingers etc.).
Some compounds which are present in living organisms specifically induce an apoptotic phenomenon. Thus, for example in mammals, the binding of the Fas ligand to the Fas membrane-bound receptor, which is also referred to as APO-1 or CD95, specifically induces an apoptosis; this apoptosis is used by the living organism to control lymphocyte populations, in particular T lymphocyte populations.
The abovementioned receptor and ligand represent an extremely advantageous physiological system which is involved in the specific elimination of cells which are no longer desired in the organism.
Mention may be made in particular of cell elimination during the maturation and activation of T lymphocytes. In fact, the Fas system, i.e. Fas ligand/Fas receptor, plays a fundamental role in the homeostasis of the immune system.
The Fas receptor is a member of a family of proteins which act as cell surface receptors and which also comprise the TNF (tumor necrosis factor) and NGF (nerve growth factor) receptors.
The Fas receptor is expressed in many cells; it is thought to accumulate in the Golgi apparatus.
The mechanism by which the Fas system induces cell death in unknown, but involves the activation of the proteases which are known under the designation ICE-like ( less than  less than interleukin-1 beta-converting enzyme greater than  greater than ) or caspases.
It may be noted that the Fas ligand can be secreted by cells in order to induce their own suicide; but given that this ligand is also found at the surface of activating cells, these cells will, as a result, induce the suicide of target cells by simple contact. Once it has been activated, the Fas receptor interacts with many intracellular proteins so as to transmit the apoptosis-triggering signal.
In vitro, other means exist for inducing apoptosis, for example by inhibiting the activity of certain kinases, and in particular kinase C; in this case, staurosporin may be used.
This product is very effective for inducing cell death by apoptosis.
It should, however, be noted that the signal transduction which is induced by staurosporin is different from that involving the Fas receptor.
However, while the means of activating apoptosis are different, the execution of the death program which is induced by these two modes of activation is equivalent and is characterized by an activation of the caspase cascade and a dysfunction of mitochondria, which releases compounds (for example, the cytochrome C) which will promote the programmed destruction of the cell. This phenomenon is energy-dependent, but does not require the synthesis of new proteins. In fact, in a cell, everything is ready for it to carry out its own destruction.
In vivo, the regulation of the apoptotic phenomenon has a considerable importance.
Specifically, many pathologies are associated with its dysfunction.
Mention may be made, for example, of two cases of apoptosis dysfunction in which the apoptosis is modified via the Fas system; they are autoimmune diseases in which apoptosis is deficient and the destruction of HIV-1-infected CD41 T lymphocytes in which apoptosis is too active.
In other cases such as the neuronal degeneration which is encountered, for example, in multiple sclerosis, apoptosis is activated via pathways which are as yet unknown.
Other pathologies exist in which apoptosis is deficient; in this respect, mention may be made of the accumulation of cancer cells in which apoptosis would appear to depend on the FAS system ( less than  less than Green greater than  greater than , Science, vol. 278, 1246, 1997).
In light of the findings recalled above, the applicant company, to its credit, found that as a result of the availability of a medicine which is capable of modifying apoptosis dysfunctions both from the point of view of an activation, in the case of the pathologies of the group comprising autoimmune diseases and the pathologies such as cancer, and from the point of view of its inhibition, in the case of the pathologies of the group comprising AIDS, it became possible to combat these diseases.
It also found, to its no less great credit, that some oligosaccharide and monosaccharide substances which optionally comprise, on at least some of their individual units, at least one substituent of the group comprising sulfate, methyl and acetyl groups, were capable of modifying apoptosis dysfunctions.
A subject of the invention is thus a medicine, characterized in that it comprises, as an active principle, an effective amount of at least one oligosaccharide substance which is capable of modifying apoptosis dysfunctions and which optionally comprises, on at least some of its individual units, at least one substituent of the group comprising sulfate, methyl and acetyl groups, said substance being chosen from the group comprising:
the oligosaccharides which are derived, by enzymatic or chemical process, from the polymers of the group comprising (1xe2x86x923)-xcex2-glucans which optionally comprise (1xe2x86x926)-xcex2-branching,
the oligosaccharides which are derived, by enzymatic or chemical process, from sulfated galactans, in particular carrageenans, agars and porphyrans.
According to one advantageous embodiment, the medicine in accordance with the invention comprises, as an active principle, an effective amount of at least one oligosaccharide which is capable of modifying apoptosis dysfunctions and which satisfies the formula:                               [                      Gluc            ⁢                                          →                                  1                  ⁢                                      xe2x80x83                                    ⁢                  3                                            β                        ⁢                          Gluc              ⁢                                                →                                      1                    ⁢                                          xe2x80x83                                        ⁢                    3                                                  β                            ⁢                              Gluc                                                                            Gluc                                                                                                                          β                        ↓                                                  1                          5                                                                                                                                                  ]                n                            (        I        )            
in which n represents an integer from 1 to 50, preferably from 5 to 10, and in which the number of branches varies from 0 to 3 per repeat unit.
According to another advantageous embodiment, the medicine in accordance with the invention comprises, as an active principle, an effective amount of at least one repeat disaccharide which is capable of modifying apoptosis dysfunctions and which satisfies the formula:                                           [                          Gal              ⁢                                                →                                      1                    ⁢                                          xe2x80x83                                        ⁢                    3                                                  α                            ⁢                              Gal                ⁢                                                      →                                          1                      ⁢                                              xe2x80x83                                            ⁢                      4                                                        β                                                      ]                    n                ⁢        Gal                            (        II        )            
in which n represents an integer from 1 to 50, preferably from 1 to 20, at least some of the repeat disaccharides of formula (II) possibly comprising one or more sulfate groups.
According to another advantageous embodiment, the medicine in accordance with the invention comprises, as an active principle, an effective amount of the product which is capable of at least partially inhibiting apoptosis and which is obtained by hydrolysis from sodium iota-carrageenate, this product consisting of a mixture of oligo-iota-carrageenans which is referred to as I9, which has a total saccharide content (determined according to Tillmans and Philippi) of 62%, and which has a distribution profile by size, which is estimated by electrophoresis on polyacrylamide gel according to Zablakis and Perez, of
The abovementioned methods are described in  less than  less than Botanica marina greater than  greater than , 33, 273-276 (1990) as regards Zablakis E. and Perez J., and in  less than  less than Biochem. Z. greater than  greater than , 215, 30-60 (1930), as regards Tillmans J. and Philippi K.
In order to prepare the product I9, the following procedure may be carried out.
The iota-carrageenan is incubated in the presence of the partially purified enzyme iota-carrageenase at a temperature of 45 to 50xc2x0 C., and then the hydrolysis products are ultrafiltered through a 10,000 Da membrane. The product I9 is thus obtained.
More particularly, the iota-carrageenan polymer is hydrolyzed with a recombinant iota-carrageenase which is overexpressed in the strain Escherichia coli. 
The preparation of the enzyme is carried out by dissolving the bacterial pellet (corresponding to 1 liter of culture) in 50 ml of 10 mM Tris pH 7.5, 100 mM NaCl, 5 mM CaCl2 buffer so as to have a final concentration of 500 U/ml.
From a practical point of view, 100 g of iota-carrageenan substrate are dissolved in 20 l of distilled water while hot (80xc2x0 C.) so as to obtain at a concentration at 5 g/l, and then the pH is adjusted to 7.5 with ammonium carbonate.
To carry out the hydrolysis, the enzyme is added to 50 U/g of polymer. The continuous ultrafiltration is begun after 30 minutes; it is a tangential ultrafiltration.
For this tangential ultrafiltration, a Pellicon machine comprising a PTGC 0.46 m2 10,000 Da cassette from the company Millipore can be used; this machine is set to 2 bar at inlet and 0.5 bar at outlet.
The filtrate outlet is partially closed in order to maintain the filtration flow rate at 1 liter per hour.
The characteristics of the reaction chamber are chosen so as to allow the enzyme to be supplied with substrate until the 20 l of solution are used up and a fixed volume of 2 liters to be maintained.
18 l are obtained of an ultrafiltrate which is concentrated to 1 liter by rotary evaporation, and then the concentrate is lyophilized. The lyophilisate thus obtained contains the product I9.
The oligocarrageenans of the I9 fraction thus obtained were subjected to an additional fractionation by low pressure chromatography on a P6 Biogel column and then on a Sephadex C10 column.
The fractions identified above are thus obtained.
According to another advantageous embodiment, the medicine in accordance with the invention comprises, as an active principle, an effective amount of the product which is capable of at least partially inhibiting apoptosis and which consists of fraction DP7 of the product I9.
According to another advantageous embodiment, the medicine in accordance with the invention comprises, as an active principle, an effective amount of the product which is capable of activating apoptosis dysfunctions, and which is obtained by acidic aqueous extraction from a brown alga named Laminaria digitata, this product consisting of a mixture of oligo-(1xe2x86x923)-xcex2-glucans which are referred to as L11 and comprise from 1 to 50, preferably from 20 to 30, saccharide units, the product in question having the NMR spectrum shown in FIG. 1.
It should be noted that the product L11 can also be obtained by aqueous extraction from brown algae in general, of which Laminaria digitata is a representative.
The preparation of the product L11 can be carried out as follows.
1 l of 0.3% sulfuric acid are gradually added to 300 g of fresh algae such as Laminaria digitata which are harvested in the month of August in fresh or dry form.
The procedure is carried out in a water bath at a temperature of approximately 70xc2x0 C. for 2 hours and 30 minutes with shaking.
This procedure is repeated twice.
The extract obtained is clarified by filtration through a filter with a porosity of 1.2 xcexcm.
The liquid resulting from this filtration is subjected to a tangential ultrafiltration through a membrane with a porosity of 50,000 daltons.
The ultrafiltration is carried out while maintaining a pressure of 1 bar.
An ultrafiltrate whose pH is brought back to 5.5, and which has a volume of approximately 0.8 liters, is thus obtained. This ultrafiltrate is subjected to dialysis on a cellulose ester membrane which has a porosity equal to 500 daltons.
A dialysate is obtained which is concentrated to a volume of 100 ml by evaporating off at 80xc2x0 C. using a Rotovapor-type machine, and then lyophilized.
7 g of a cream-colored powder which constitutes the product L11 are obtained.
Analysis by ionic chromatography coupled with amperometry and using an ion-exchanging resin sold by the company Dionex shows that the oligo-(1xe2x86x923)-xcex2-glucans which are constituents of the abovementioned powder in fact have 1 to 50, preferably from 20 to 30, saccharide units.
With the chromatographic conditions (so-called HPLC method, i.e.  less than  less than High pressure liquid chromatography greater than  greater than ) being as follows:
the curve which is shown in FIG. 16 was obtained which identifies the product L11.
The examination of the 13C NMR spectrum of the product L11, which was carried out using an 80 mg/ml solution in D2O and which is represented in FIG. 1, shows a (1xe2x86x923)-xcex2-D-glucan backbone for which the resonances of the various carbons were able to be identified (they are assembled in Table A below) by comparison with the values in the literature [see Williams et al., 1992  less than  less than Development of a water soluble, sulfated (1xe2x86x923) xcex2 D-glucan biological response modifier derived from Saccharomyces cerevisiae greater than  greater than , Carbohydr. Res. 23b: 247:25].
The medicines in accordance with the invention which are defined above comprise the adjuvants of conventional formulation corresponding to their mode of administration and dose used.
A subject of the invention is also a method for preparing a medicine for treating apoptosis dysfunctions, characterized in that a pharmaceutical composition comprises at least one of the active principles identified above.
According to one advantageous embodiment, the abovementioned pharmaceutical composition is suitable for intravenous administration.